Dual Small-Molecule Targeting of SMAD Signaling Stimulates ...€¦ · toward Neural Lineages Methichit Wattanapanitch1, Nuttha Klincumhom1, Porntip Potirat1, ... remarkably expressed

Dual Small-Molecule Targeting of SMAD SignalingStimulates Human Induced Pluripotent Stem Cellstoward Neural LineagesMethichit Wattanapanitch1, Nuttha Klincumhom1, Porntip Potirat1, Rattaya Amornpisutt1,

Chanchao Lorthongpanich1, Yaowalak U-pratya2, Chuti Laowtammathron1, Pakpoom Kheolamai3,

Niphon Poungvarin4, Surapol Issaragrisil1,2*

1 Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, 2Division of Hematology, Department of

Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, 3Division of Cell Biology, Department of Pre-clinical Sciences, Faculty of Medicine,

Thammasat University, Pathumthani, Thailand, 4Division of Neurology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok,

Thailand

Abstract

Incurable neurological disorders such as Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD)are very common and can be life-threatening because of their progressive disease symptoms with limited treatmentoptions. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurologicaldiseases, we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiatedthem into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiencywas improved by supplementing the histone deacethylase inhibitor, valproic acid (VPA), and inhibitor of p160-Rhoassociated coiled-coil kinase (ROCK), Y-27632, after retroviral transduction. We obtained a number of iPS colonies thatshared similar characteristics with human embryonic stem cells in terms of their morphology, cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. Aftertreatment with Noggin and SB431542, inhibitors of the SMAD signaling pathway, HDF-iPSCs demonstrated rapid andefficient differentiation into neural lineages. Six days after neural induction, neuroepithelial cells (NEPCs) were observed inthe adherent monolayer culture, which had the ability to differentiate further into NPCs and neurons, as characterized bytheir morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be appliedto generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drugsensitivity assays.

Citation: Wattanapanitch M, Klincumhom N, Potirat P, Amornpisutt R, Lorthongpanich C, et al. (2014) Dual Small-Molecule Targeting of SMAD SignalingStimulates Human Induced Pluripotent Stem Cells toward Neural Lineages. PLoS ONE 9(9): e106952. doi:10.1371/journal.pone.0106952

Editor: Irina Kerkis, Instituto Butantan, Brazil

Received June 3, 2014; Accepted August 1, 2014; Published September 10, 2014

Copyright: � 2014 Wattanapanitch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and itsSupporting Information files.

Funding: This study was supported by grant from the Thailand Research Fund (RTA 488-0007 to S.I. and DBG no. 4980014 to N.P.), and the Commission onHigher Education (grant no. CHE-RES-RG-49 to S.I.). S.I. is a Senior Research Scholar of Thailand Research Fund. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* Email: [email protected]

Introduction

Neurological disorders including Parkinson’s disease (PD),

Huntington’s disease (HD) and Alzheimer’s disease (AD) are

considered to be irreversible and incurable due to progressive

neural loss and dysfunction [1]. Although neural stem cells (NSCs)

in the brain can be activated to proliferate and differentiate to

mature neurons, which can migrate toward the site after neural

degeneration or injury, the number of neural cells normally

derived from endogenous NSCs appears inadequate for the

replacement of neural loss [2]. Various cell sources both from fetal

and adult NSCs have been applied to neural transplantation but

their limited proliferation capacity in vitro has hindered clinical

applications [3].

Over the last decade, human embryonic stem cells (hESCs) have

provided a great promise not only as an unlimited renewable

source of surrogate cells to repair damaged tissues, but also as a

model to study embryonic development and disease mechanisms.

Nevertheless, the derivation of hESCs requires human oocytes and

subsequent destruction of human embryos, which raise significant

ethical concerns. Recent advances in somatic cell reprogramming

have provided unlimited numbers of patient-specific pluripotent

stem cells [4,5]. The induced pluripotent stem cells (iPSCs) are

comparable to hESCs in terms of their self-renewal and

differentiation potential without the ethical issues and immuno-

logical rejection when used for autologous transplantation.

Several attempts have been made to differentiate human

pluripotent stem cells (hPSCs) to neural progenitor cells (NPCs),

which can differentiate further to all neural subtypes including

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neurons and glial cells [6]. The common neural differentiation

protocol has been demonstrated by the formation of embryoid

bodies (EB), which is simple, cost-effective and scalable, but

heterogeneous cell populations are also generated within the EBs

[7]. Co-culturing with mouse mesenchymal stromal cell lines such

as PA6 and MS5 cells has been demonstrated to induce neural

differentiation by their secretory factors. However, the clinical

application of this method has been impeded by the risk of animal

cell contamination and by the fact that the secretory factors at play

are undefined [8].

To overcome these limitations, a differentiation strategy using

serum-free defined factors is essential [9]. Using the knowledge of

factors and signaling pathways involving in fetal neural develop-

ment, hPSCs can be induced to efficiently differentiate into neural

lineages. Several studies in Xenopus laevis indicated that the

inhibitors of bone morphogenetic protein (BMP) including

Noggin, Follistatin and Chordin play an important role during

neural development of embryo through the SMAD signaling

pathway [10–12]. In adult mouse brain, Noggin has been

demonstrated to be an essential neural-inducing factor and

remarkably expressed in nervous system [13]. The addition of

recombinant Noggin improved the efficiency of neural conversion

of hESCs in culture [14]. Previously, a small molecule, SB431542,

has been shown to enhance the neural differentiation of hPSCs

through the inhibition of transforming growth factor-beta (TGFb)pathway, which results in the downstream inhibition of SMAD

signaling [15]. The synergistic action of Noggin and SB431542 has

been shown to rapidly drive cell fate alteration from pluripotent to

NPC stage, which can be further committed to specific neural cell

types such as cortical neurons [16,17]. This strategy could thus

enhance the potential use of iPSC-derived neurons in future

clinical applications.

The present study aims to establish a protocol for iPSC

generation and differentiation to NPCs and mature neurons

through dual-action of small molecules during neuronal induction

period. This rapid and efficient differentiation strategy could be

further used for the generation of patient-specific iPSC lines from

patients’ fibroblasts with several neurological diseases and would

provide an alternative source of pluripotent stem cells for the study

of molecular mechanisms, early embryonic developmental path-

ways [18], the pathological basis of genetic disorders as well as

toxicology or pharmacology testing upon neuronal lineage

differentiation in future studies [19,20].

Materials and Methods

Cell cultureHuman dermal fibroblasts (HDFs) (ScienCell, USA) and human

foreskin fibroblasts (HFFs) (ATCC) were maintained in fibroblast

medium: DMEM supplemented with 10% fetal bovine serum

(FBS) (Lonza, Switzerland), 1x GlutaMAX and 25 U/ml penicil-

lin, 25 mg/ml streptomycin. The hESC line (Chula2.hES) [21]

and iPSCs were maintained on c-irradiated (45 Gy) HFF (iHFF)

and cultured in hESC medium, which contains knockout

Dulbecco’s modified Eagle’s medium (KO-DMEM)-high glucose,

20% knockout serum replacer, 2 mM L-glutamine, 0.1 mM non-

essential amino acids, 0.1 mM 2-mercaptoethanol, 1x insulin-

transferrin-selenium, 25 U/ml penicillin, 25 mg/ml streptomycin

and 4 ng/ml basic fibroblast growth factor (bFGF). hESCs and

iPSCs were subcultured approximately once every 5 days by

incubation with 0.05% Trypsin for 2 min as described previously

[22] (all reagents were from Invitrogen, USA). Alternatively,

hESCs and iPSCs were maintained in mTESR1 medium (Stem

Cell Technologies, Canada) on Matrigel (BD Bioscience, USA)-

coated cell culture plates. hESCs and iPSCs were subcultured

approximately once every 5 days by incubation with 1 mg/ml

Dispase (Stem Cell Technologies, Canada) for 7 min as per

standard hESC procedures.

Generation of induced pluripotent stem cellsMoloney-based retroviral vectors (pMXs) containing human

complementary DNAs (cDNAs) of OCT4, SOX2, KLF4 and C-

MYC (Cell Biolabs, USA) were transfected into a retroviral

packaging cell line, Platinum-A cells (Cell Biolabs) using FuGENE

HD transfection reagent (Promega Corporation, USA). One day

before infection, passages 6–8 of the HDF were seeded at 100,000

cells per well of 6-well plate. The viral supernatant from each plate

was collected 48 h post-transfection and the plates were replaced

with fresh medium for consecutive infections. The viral superna-

tant was filtered through a 0.45 mm pore-size filter and

supplemented with 4 mg/ml polybrene before infecting HDFs.

For experimental control, we transfected Platinum-A cells with

pMX-GFP retroviral vector (Cell Biolabs) and transduced HDFs

with the viral supernatant. The cells were infected 3 times at 12 h

intervals. After infection, 0.5 mM valproic acid (VPA) (Merck,

USA) was supplemented to the culture for the first 10 days to

optimize the colony formation efficiency [23]. On day 4 post-

infection, these cells were treated with 10 mMY-27632 for an hour

prior to trypsinization and transferred onto iHFF in hESC

medium supplemented with 0.5 mM VPA and 10 mM Y-27632

(Merck, USA). The cells were cultured until ES-like colonies

appear. These colonies were picked and transferred to a fresh

iHFF feeder plate for expansion (Figure 1A). The iPSC lines were

named HDF-iPSCs and adapted to feeder-free system for further

expansion and characterization.

In vitro differentiation of human iPSCsSpontaneous differentiation via embryoid body (EB) formation

was performed by treating HDF-iPSCs with 1 mg/ml Dispase for

30 min until colonies lifted off the plate. The colonies were washed

in KO-DMEM basal medium and transferred to low attachment

dish in hESC medium without bFGF supplementation for a week.

The EBs were then seeded onto 0.1% gelatin-coated dishes and

cultured in the same medium for another 3 weeks. Lineage marker

gene and protein expression were examined by quantitative

reverse transcription polymerase chain reaction (RT-qPCR) and

immunofluorescent staining, respectively.

In vitro neural differentiation of human iPSCsHDF-iPSCs were induced to differentiate into neuronal lineages

as adherent monolayer culture system as previously described with

modifications [24]. Briefly, HDF-iPSCs were dissociated into

single cells with Accutase. The cells were seeded at a density of

3.5–56104 cells/cm2 in mTESR1 medium on Matrigel-coated

plates in order to obtain 100% confluency within one day after

seeding. Thereafter, the medium was changed to neural induction

medium, which contains a 1:1 mixture of DMEM/F12 glutamax

and neurobasal medium, 1x N2 supplement, 1x B27 supplement,

5 mg/ml insulin, 1 mM L-glutamine, 0.1 mM non-essential amino

acids, 0.1 mM 2-mercaptoethanol, 25 U/ml penicillin, 25 mg/ml

streptomycin, and supplemented with 200 ng/ml Noggin (R&D

system, USA) and 10 mM SB431542 (Tocris Bioscience, USA).

The medium was changed every day for 6 days. The neuroep-

ithelial cells (NEPCs) were dissociated with 1 mg/ml Dispase for

3 min and triturated into small clumps. The cells were replated

onto laminin-coated plates in neural induction medium. On day 7

of differentiation, the NEPCs were cultured in the neural

induction medium minus Noggin and SB431542. The medium

Neural Differentiation of hiPSCs through SMAD Inhibitors


was supplemented with 20 ng/ml bFGF for 3–4 days for neural

rosette formation. Mature neuronal differentiation was further

performed by bFGF removal and cultured on polyornithine and

laminin-coated plates. The medium was changed every 2–3 days.

Gene expression analysis by RT-qPCRTotal RNA was prepared using TRIzol reagent (Invitrogen

Corporation). cDNA was prepared using 2 mg of RNA and reverse

transcribed using SuperScript III First-Strand Synthesis System

Figure 1. Generation and characterization of iPSCs from human dermal fibroblasts (HDFs). (A) Schematic diagram represents a protocolto generate iPSCs from HDFs by retroviral transduction. (B) Morphology of HDFs on day 1 post-transduction (i), non ESC-like colonies on day 8 (ii),ESC-like colonies on day 13 (iii), iPS colonies on iHFF plate (iv) and an iPS colony on Matrigel-coated plate (v), scale bar (i) and (iii) = 200 mm, (ii), (iv)and (v) = 500 mm. (C) RT-qPCR analysis of pluripotency- and fibroblast-associated genes in HDF, HDF-iPSC1 and Chula2.hES cells. (D) RT-PCR analysisof pluripotent genes, OCT4 and NANOG, of all the established iPS clones, HDF-iPSC2-6, as compared to Chula2.hES cells. (E) Representativeimmunofluorescent images of pluripotent transcription factors and cell surface antigens of HDF-iPSC1 as compared to Chula2.hES cells, scalebar = 100 mm.doi:10.1371/journal.pone.0106952.g001



and Oligo (dT) primers (Invitrogen Corporation). qPCR analysis

was carried out on 7500 Fast Real-time PCR machine (Applied

Biosystems, California) using Power SYBR Green PCR Master

Mix (Applied Biosystems). The PCR reaction consisted of 1x

SYBR Green PCR master mix, 200 nM of forward and reverse

primers, 1 ml of template cDNA. The total volume was adjusted to

20 ml with nuclease-free water. Initial enzyme activation was

performed at 95uC for 10 min, followed by 40 cycles of

denaturation at 95uC for 15 s, and primer annealing/extension

at 60uC for 1 min. Melting curve analysis was performed to

determine primer specificity. The relative expression of each gene

was normalized against the house-keeping gene product, Glycer-

aldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequenc-

es are provided in Table S1 (Supplementary data). Each

experiment was carried out 3 times.

Immunofluorescent stainingCells were washed with phosphate buffered saline (PBS) and

fixed with 4% paraformaldehyde for 20 min. After washing with

PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma) in

PBS for 15 min for intracellular staining. Cells were then blocked

with 3% bovine serum albumin (BSA) (Sigma) in PBS for 1 h at

room temperature and incubated with primary antibodies diluted

in 1% (w/v) BSA in PBS overnight at 4uC. Cells were washed with

PBS three times and stained with secondary antibodies for 1 h at

room temperature in the dark. The following primary and

secondary antibodies were used: mouse anti-human OCT4

(AbD Serotec, UK), rabbit anti-human NANOG (Millipore),

Alexa Fluor 488 anti-human TRA-1-60 antibody (BioLegend),

mouse anti-human TRA-1-81 (Millipore), mouse anti-human

Stage-Specific Embryonic Antigen-4 (SSEA-4) (Millipore), mouse

anti-human NESTIN (Millipore), rabbit anti-human TUJ1

(Covance), mouse anti-human a-fetoprotein (Calbiochem), mouse

anti-human actin (muscle) clone HHF35 (AbD Serotec), mouse

anti-human PAX6 (Millipore), Alexa Fluor 488 coat anti-mouse

IgG (H+L), Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa

Fluor 568 goat anti-mouse IgG (H+L) (Life Technologies). Nuclei

were stained with prolong gold DAPI with antifade (Invitrogen).

Cells were visualized with an Eclipse TE2000 fluorescent

microscope (Nikon, Japan).

Teratoma formationAll animal experiments were performed with an approval of

Siriraj Animal Care and Use Committee (SiACUC), number 002/

2556. Briefly, HDF-iPSCs were treated with 10 mM of Y-27632

for 1 h prior to harvesting with accutase. Cells were resuspended

at 16107 cells/200 ml of cold 30% (v/v) Matrigel in KO-DMEM

basal medium and implanted intramuscularly into 6–8 weeks old

nude (Foxn1nu) mice obtained from National Laboratory Animal

Center, Mahidol University. Chula2.hES cells were also injected

into nude mice as a positive control. Teratomas were removed 8

weeks post-transplantation, fixed in 10% neutral buffered formalin

and embedded in paraffin wax. Samples were sectioned and

examined by hematoxylin and eosin (H&E) staining.

Karyotypic analysisChromosomal analysis was performed at Siriraj Central

Cytogenetic Laboratory, Faculty of Medicine Siriraj Hospital,

Mahidol University. Briefly, HDF-iPSCs were cultured in T-25

flask on Matrigel in mTESR1 medium for 5 days until reaching

80% confluency. Cells were treated with colcemid to block the cell

division at metaphase stage and harvested for analysis using a

standard protocol for G-banded karyotyping.

Statistical analysisData are presented as mean 6 SD from three independent

experiments. The statistics generated in this study were performed

using GraphPad Prism 5 (GraphPad Software, Inc).

Results

Generation of human iPSCs from human dermalfibroblastsWe generated human iPSCs using retroviral transduction with 4

vectors encoding human cDNA of OCT4, SOX2, KLF4 and C-

MYC into human dermal fibroblasts (Figure 1A). On day 4 post-

infection, the infected cells were transferred to iHFF plates. Non

ESC-like colonies with cobblestone phenotype appeared as early

as day 8 post-infection (Figure 1B-ii). We observed small ESC-like

colonies from days 13–28 post-infection (Figure 1B-iii). More than

30 iPS colonies were observed from 2 wells of a 6-well plate in one

experiment. Those colonies were physically dissected and trans-

ferred to iHFF feeder plates for expansion. After 3 passages, the

cells were adapted to feeder-free condition. The HDF-iPS colonies

were compact with clearly defined edges on both iHFF feeders and

feeder-free conditions (Figure 1B-iv and v, respectively).

HDF-iPSCs display typical characteristics of hESCsWe isolated a total of 6 clones (HDF-iPSC1-6); one clone (HDF-

iPSC1) was fully characterized. The undifferentiated HDF-iPSC1

cell line typically expressed endogenous pluripotency-associated

genes including NANOG, OCT4, SOX2, GDF3, REX1 and

TERT at similar levels to those of Chula2.hES cells as shown by

RT-qPCR. These markers were markedly upregulated as com-

pared to those of the parental HDF. Expression of fibroblast-

specific markers including HOXA9 and ZFPM2 were downreg-

ulated as compared to those of the HDF (Figure 1C). Other iPS

clones, HDF-iPSC2-6, also expressed OCT4 and NANOG as

determined by standard RT-PCR (Figure 1D). Moreover, the

HDF-iPSC1 cells also expressed transcription factors, OCT4 and

NANOG, and specific cell surface antigens, SSEA-4, TRA-1-60

and TRA-1-81, with a similar pattern to those observed in

Chula2.hES cells, as demonstrated by immunofluorescent staining

(Figure 1E).

The in vitro pluripotency of HDF-iPSC1 cells was assessed by

spontaneous differentiation via embryoid body (EB) formation.

The HDF-iPSC1 cells formed EBs in suspension culture in a

similar manner to hESCs. The EBs were then seeded onto gelatin-

coated plate where the cells were seen to differentiate to neurons,

adipocytes and beating cardiomyocytes (Figure 2A and Movie S1).

Expression of pluripotency marker genes, NANOG and OCT4,were downregulated upon differentiation whereas the lineage-

specific marker genes, SOX1 (ectoderm), FLK1 (mesoderm),

FOXA2 and AFP (endoderm) were upregulated after 4 weeks of

differentiation, when compared to undifferentiated HDF-iPSC1 or

Chula2.hES cells (Figure 2B). The differentiated HDF-iPSC1 cells

also expressed markers of three embryonic germ layers including

AFP (endoderm), SMA (mesoderm) and NESTIN (ectoderm) as

shown by immunofluorescent staining (Figure 2C).

We then examined the pluripotency of HDF-iPSC1 cells in vivoby injecting 16107 cells intramuscularly into nude mice.

Histological analysis at 8 weeks post-transplantation revealed that

the HDF-iPSC1-derived teratomas consisted of tissues represent-

ing all 3 embryonic germ layers including neural rosettes/

epithelium (ectoderm), cartilage tissue (mesoderm) and glandular

tissue (endoderm) (Figure 2D). Karyotyping of cultured HDF-

iPSC1 cells at passage 32 was analyzed by chromosomal counting

and G-banded karyotyping. The results from 25 metaphase-stage



cells indicated that the HDF-iPSC1 cells were karyotypically

normal (46, XY) (Figure 2E).In vitro differentiation of HDF-iPSC1 to neural lineagesWe generated NPCs of the cerebral cortex from HDF-iPSC1

cells in monolayer culture using dual SMAD signaling inhibitors

(Figure 3A). HDF-iPSC1 cells were induced to differentiate into

Figure 2. In vitro and in vivo differentiation, and karyotypic analysis of HDF-iPSC1 cells. (A) Representative micrographs show morphologyof the embryoid body (EB) of HDF-iPSC1 cells on day 1 in suspension culture (i), differentiated EB turned into neurons (ii), adipocytes (iii) and beatingcardiomyocytes (iv), scale bar (i) = 500 mm, (ii) and (iii) = 50 mm and (iv) = 200 mm. (B) RT-qPCR analysis of pluripotent and lineage markers of thedifferentiated EB generated from HDF-iPSC1 and Chula2.hES cells as compared to the undifferentiated HDF-iPSC1 and Chula2.hES cells. (C)Immunofluorescent analysis showed the expression of lineage markers, AFP (endoderm), SMA (mesoderm) and NESTIN (ectoderm) of the EBoutgrowth generated from HDF-iPSC1 cells after 4 weeks of differentiation, scale bar (i–iv) = 50 mm, (v) and (vi) = 100 mm. (D) Hematoxylin and eosin(H&E) staining of teratomas derived from HDF-iPSC1 cells at 8 weeks post-implantation into nude mice. The teratomas contained heterogeneoustissues of all three embryonic germ layers including neural rosettes (ectoderm), cartilage (mesoderm) and glandular structure (endoderm). (E)Representative karyotype analysis of HDF-iPSC1 cells at passage 32 shows normal karyotype (46, XY).doi:10.1371/journal.pone.0106952.g002



NEPC stage, which was identified by their morphology showing a

tightly packed neuroepithelial sheet with small nuclei (Figure 3B-

iii). To select an appropriate timing for NEPC generation, we

examined the expression levels of NEPC marker, PAX6, on days

4, 5 and 6 of neural induction by immunofluorescent staining. The

number of PAX6-positive cells gradually increased and was

highest on day 6 of differentiation (Figure S1 and Figure 3B-iv).

This pattern of PAX6 expression correlated with data from RT-

qPCR analysis on days 4 and 6 of differentiation, which

demonstrated that the expression level of OCT4 was downregu-

lated and that of PAX6 was upregulated on day 6 (Figure S2). The

expression level of neuroepithelial marker gene, OTX1, was also

upregulated after 6 days of differentiation when compared with

undifferentiated HDF-iPSC1 cells (Figure 3C).

Thereafter, we induced NEPCs to differentiate to NPCs, which

is radially organized as a neural rosette structure. These iPSC-

derived NPCs were expanded in monolayer culture using bFGF

supplementation and expressed the transcription factor, NESTIN,

as demonstrated by immunofluorescent staining (Figure 3B-v).

After bFGF removal, a majority of NPCs were further differen-

tiated to post-mitotic mature neurons, which exhibited neural

outgrowth and formed neural network, as characterized by

immunofluorescent staining with TUJ1 (Figure 3B-vi). Likewise,

RT-qPCR analysis demonstrated that the expression levels of

Figure 3. Neural differentiation of HDF-derived iPSCs and characterization of differentiated neurons. (A) Schematic diagram representsthe strategy to differentiate HDF-derived iPSCs into neural lineages using SMAD signaling inhibitors. (B) Morphological changes in HDF-iPSC1 culturesduring neural differentiation; undifferentiated HDF-iPSC1 cells on day -1 (i), differentiated cells on day 2 (ii) and day 6 (iii), representativeimmunofluorescent images show the NEPC on day 6 (iv), NPC on day 9 (v) and post-mitotic neuron on day 28 (vi) of differentiation as examined byPAX6, NESTIN and TUJ1 markers, respectively. Cell nuclei were stained with DAPI. Scale bar = 100 mm. (C) RT-qPCR analysis of pluripotency- andneural-associated genes in NEPCs on day 6 and mature neuron on day 28 of differentiation as compared to those of undifferentiated HDF-iPSC1 cells(arbitrarily set at 1).doi:10.1371/journal.pone.0106952.g003



neural-specific marker genes, TUJ1 and MAP2, were significantlyupregulated as compared to those of the undifferentiated HDF-

iPSC1 cells (Figure 3C).

Discussion

The objective of our study was to develop a strategy to generate

human iPSC-derived neural cell lineages to serve as an unlimited

renewable source for repair of damaged tissues and also as in vitromodels in which to study the underlying mechanisms of

neurological diseases. The reprogramming efficiency was en-

hanced by transducing the HDFs with viral supernatant three

times every 12 hours and supplementing the transduced cells with

the histone deacetylase inhibitor, VPA, for 10 days post-infection.

VPA has been shown to be the most potent chemical for

augmenting the reprogramming efficiency of mouse embryonic

fibroblasts and human fibroblasts compared with other histone

deacetylase inhibitors, such as suberoylanilide hydroxamic acid

(SAHA) and trichostatin A (TSA), and DNA methyltransferase

inhibitor, 59-azacytidine [23,25]. We observed a greater number

of iPS colonies in the transduced fibroblasts, which were

supplemented with VPA, when compared to those without VPA

(data not shown). In addition, before transferring the transduced

HDFs to a new iHFF feeder plate, we supplemented the culture

with Y-27632, an inhibitor of p160-Rho associated coiled-coil

kinase (ROCK), which has been shown to prevent dissociation-

induced apoptosis in several studies [22,26–29]. The use of

ROCK inhibitor improved iPSC survival and adherence to the

feeder layer. The established HDF-iPSCs shared similar charac-

teristics with Chula2.hES line, in terms of their morphology, cell

surface antigens, pluripotency-associated gene and protein expres-

sions as well as the in vitro differentiation potential to generate

derivatives of three embryonic germ layers.

This study also demonstrates the neural differentiation of HDF-

derived iPSCs on adherent monolayer culture with defined factors.

Our optimized culture condition could induce HDF-iPSCs to

differentiate to NEPCs, NPCs and mature neurons. By using a

combination of Noggin and SB431542, HDF-iPSC1 cells rapidly

lost their pluripotency and became committed to a neural cell fate,

which can be observed from day 4 after induction. Our result was

consistent with the previous study, which demonstrated that dual

SMAD inhibition greatly induced loss of OCT4 expression [16]. In

contrast to previous studies where neural induction was performed

for 8–12 days prior to neural rosette formation [16,17,24], in our

system, the differentiated cells cultured beyond 6 days were

densely packed and started to detach at the edge of culture plate

(data not shown). Therefore, we performed NEPC induction for 6

days and observed the confluent homogenous neuroepithelial

sheet of the differentiated cells. These cells progressively lost their

OCT4 expression, strongly expressed PAX6 and demonstrated

their ability to differentiate further into NPCs after NEPC

dissociation.

Several studies reported the application of these inhibitors in

neural differentiation protocols, either alone or by dual supple-

mentation. The BMP inhibitor, Noggin was previously shown to

induce neural differentiation of hESCs under culture conditions

using defined factors [30]. By adding Noggin, neurospheres can

potentially be generated from hESCs by suppressing the expres-

sion of endodermal and mesodermal cell fates in suspension

culture [31] while the addition of SB431542, a selective inhibitor

of the TGFb pathway, inhibits SMAD signaling by modulating

phosphorylation of ALK4, ALK5 and ALK7 receptors [32].

SB431542 mediated p-Erk(ERK)1/2 signaling that promotes

neural conversion, which results in increased NPCs derived from

mESCs and hESCs through inhibition of the Activin/ALK4

pathway [33]. Taken together, the combination of Noggin and

SB431542 thus endowed a synergistic effect on cell fate alteration

from pluripotent stem cells to neural lineages [17]. These dual

SMAD inhibitors have been used for several neural differentiation

strategies to derive NPCs that can give rise to specific neural cell

types such as cortical and dopaminergic neurons [16,34].

Therefore, these iPSC-derived NPCs may be considered as an

unlimited renewable cell source for neurological disease modeling

and for cell-based transplantation.

Recently, in vitro models of neurological diseases have been

utilized to uncover the underlying disease mechanisms. Derivation

of AD patient-specific iPSCs has provided a model for studying

pathogenesis and drug discovery [35]. The isogenic PD patient-

derived iPSCs have been successfully generated for modeling

genetic and molecular mechanisms of the disease in which the

specific pathway was discovered for developing targeted therapy

for PD [36]. Moreover, the cytosine adenine guanine (CAG)

repeat iPSC lines were established from HD followed by successful

targeted gene correction of HD by homologous recombination.

The genetically corrected iPSCs gave rise to specific neural

subtypes, which reversed disease phenotype of the neural stem

cells and could potentially be used for cell replacement therapy in

the future [37].

In conclusion, the present study provides a protocol to generate,

characterized human iPSC lines from HDF and further differen-

tiate them to mature neurons using dual SMAD signaling

inhibitors. The approach described in this study can be used to

generate patient-specific PSCs, which can be used to study

mechanisms underlying patient’s neurological disease. When

combined with an efficient gene correction, these genetically

corrected iPSCs can provide an autologous cell source for cell

replacement therapy.

Supporting Information

Figure S1 Representative immunofluorescent imagesshow PAX6-positive cells on days 4, 5 and 6 of neuralinduction.(TIF)

Figure S2 RT-qPCR analysis of OCT4 and PAX6 ofdifferentiated cells on days 4 and 6 of neural inductionas compared to those of undifferentiated HDF-iPSC1cells.(TIF)

Table S1 Primer sequences for RT-qPCR.(DOCX)

Movie S1 Beating cardiomyocytes from differentiatedEBs of HDF-iPSC1 cells.(M4V)

Acknowledgments

We thank Barbara Knowles for critical review of this manuscript.

Author Contributions

Conceived and designed the experiments: MW NK. Performed the

experiments: MW NK PP RA C. Lorthongpanich. Analyzed the data: MW

NK PP RA C. Lorthongpanich YU C. Laowtammathron PK. Contributed

reagents/materials/analysis tools: NP SI. Contributed to the writing of the

manuscript: MW NK SI.



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